Microglia and Opioid Withdrawal: Mechanisms of Negative Reinforcement

目前阿片类药物过量的流行是由非法和处方麻醉性疼痛推动的。 药物。阿片类药物的广泛使用和反复戒断增加了严重戒断和 通过消极强化的方式使复发的脆弱性永久化。消极的情感价值 退缩可能会在最初戏剧性的身体迹象之后很长时间持续，包括长期的负面情绪 国家，对毒品的渴望，以及很高的复发可能性。这些综合症状通常被称为 “吸毒成瘾”。上瘾的人往往更愿意继续吸毒，而不是面临戒断。存在 “吸毒成瘾”具有严重炎症状态的许多特征，这导致我们调查了 小胶质细胞，驻留在大脑中的先天免疫细胞，参与阿片类药物的耐受和戒断，这是 由CEBRA R21赠款(R21-DA044757)支持。 CEBRA R21拨款导致我们发现核糖体结合的mRNAs发生了戏剧性的变化- “翻译组”--在小胶质细胞中使用核糖核酸标签纯化的小胶质细胞RNA测序。那些结果 为我们提供了构成这项提议基础的线索。许多变化与环磷酸腺苷有关 信号及其下游靶点，以及Gi/o信号的实验性化学发生刺激 实际上会加重阿片类药物的戒断。了解到神经胶质细胞是可塑性的伙伴，我们怀疑 阿片类药物依赖的复发/缓解特性有助于启动和调节小胶质细胞，从而转移影响 从初期温和戒断到加剧戒断和多次寻求阿片类药物 容忍和退缩的循环。因此，对神经胶质细胞在戒断中的作用的研究可能会提供新的 治疗途径。我们提出了使用芬太尼和最近开发的转基因小鼠的三个目的 允许条件性和小胶质细胞特异性的Cre和TdTomato表达，而不会中断小胶质细胞的功能。在……里面 目的1我们将分析小胶质细胞翻译组在阿片类药物1个周期和5个周期后的变化轨迹 依赖和自发戒断。在目标2中，我们将检查身体和行为 阿片类药物依赖和戒断的一个周期和五个周期的后果，以探索间歇性 依赖和退出的循环加剧了退出的负面后果。到时候我们会的 探讨嘌呤能受体P2Y12和P2X7参与小胶质细胞反应的假说 在阿片类药物戒断的初始和延迟阶段。在目标3中，我们将使用体外脑片模型，其中2- 小胶质细胞的光子共聚焦成像。我们将利用时间来研究小胶质细胞的微结构和运动性- 失活显微镜。我们将使用基于FRET的生物传感器和 GCaMP6的钙动力学。这三个目标整合了时间、行为和分子 阿片类药物依赖和戒断期间小胶质细胞参与的后果。

The current epidemic of opioid overdoses has been propelled by both illicit and prescribed narcotic pain medications. Extensive opioid use and repeated abstinence increases the likelihood of severe withdrawal and perpetuates the vulnerability to relapse via means of negative reinforcement. The negative emotional valence of withdrawal can last long after the initial, dramatic physical signs, involving a protracted negative emotional state, drug craving, and a high likelihood of relapse. These combined symptoms are commonly referred to as being “dope sick”. Addicted individuals often prefer to continue drugs rather than face withdrawal. Being “dope-sick” has many attributes of a severe inflammatory state and this led us to investigate the involvement of microglia, the innate immune cells that reside in the brain, in opioid tolerance and withdrawal, and this was supported by a CEBRA R21 grant (R21-DA044757). That CEBRA R21 grant resulted in our findings of dramatic changes in ribosome-bound mRNAs—the “translatome”—in microglia using RNA sequencing of RiboTag purified microglial RNAs. Those results provided us with the leads that form that basis for this proposal. Many of the changes related to cyclic AMP signaling and its downstream targets, and experimental chemogenetic stimulation of Gi/o signaling was found to actually worsen opioid withdrawal. With the understanding that glia are partners in plasticity, we suspect that the relapsing/remitting nature of opioid dependence serves to prime and condition microglia, shifting the impact from tempering withdrawal during initial stages to exacerbating withdrawal and opioid seeking after multiple cycles of tolerance and withdrawal. Thus, investigations into the role of glia in withdrawal may provide new therapeutic avenues. We propose three Aims using fentanyl and a recently developed transgenic mouse that allows conditional and microglia-specific Cre and TdTomato expression without disrupting microglia function. In Aim 1 we will analyze the trajectory of the changing microglial translatome after one vs. five cycles of opioid dependence and spontaneous withdrawal. In Aim 2 we will examine the physical and behavioral consequences of one vs. five cycles of opioid dependence and withdrawal, to explore the idea that intermittent cycles of dependence and withdrawal exacerbate the negative consequences of withdrawal. We will then investigate the hypothesis that the purinergic receptors P2Y12 and P2X7 are involved in microglial responses during initial and delayed phases of opioid withdrawal. In Aim 3 we will use an in vitro brain slice model with 2- photon confocal imaging of microglia. We will study the microstructure and motility of microglia using time- lapse microscopy. We will measure real-time changes in cyclic AMP using a FRET-based biosensor and calcium dynamics with GCaMP6. These three Aims integrate the temporal, behavioral, and molecular consequences of microglial engagement during opioid dependence and withdrawal.